Resveratrol supplementation at old age reverts changes associated with aging in inflammatory, oxidative and apoptotic markers in rat heart.

PURPOSE: Aging is known to play a critical role in the etiopathogenesis of several diseases. Among them, cardiovascular disorders are especially relevant since they are becoming the first cause of death in western countries. Resveratrol is a polyphenolic compound that has been shown to exert beneficial effects at different levels, including neuronal and cardiovascular protection. Those effects of resveratrol are related, at least in part, to its antioxidant and anti-inflammatory properties. In the current investigation we were interested in exploring whether the positive effects of resveratrol at cardiac level were taking place even when the supplementation started in already old animals. METHODS: Old male rats were supplemented with resveratrol during 10 weeks. Using RT-PCR, we analyzed the effects of resveratrol supplementation on the expression of different genes related to inflammation, oxidative stress and apoptosis in rat heart. RESULTS: Resveratrol reverted age-related changes in inflammatory, oxidative and apoptotic markers in the rat heart. Among others, the expression of two major inflammatory markers, INF-γ and TNF-α and two oxidative markers, heme oxygenase-1 and nitric oxide synthase, were increased with aging, and resveratrol supplementation reduced the level of some of these to those observed in the heart of young animals. Moreover, age-related changes in apoptotic markers in rat heart tend to be also reverted by resveratrol treatment. CONCLUSION: Our results suggest that resveratrol might exert beneficial effects as an anti-aging compound to revert age-related changes in cardiac function.

Mitochondrial base excision repair positively correlates with longevity in the liver and heart of mammals.

Damage to DNA is especially important for aging. High DNA repair could contribute, in principle, to lower such damage in long-lived species. However, previous studies showed that repair of endogenous damage to nuclear DNA (base excision repair, BER) is negatively or not correlated with mammalian longevity. However, we hypothesize here that mitochondrial, instead of nuclear, BER is higher in long-lived than in short-lived mammals. We have thus measured activities and/or protein levels of various BER enzymes including DNA glycosylases, NTHL1 and NEIL2, and the APE endonuclease both in total and mitochondrial liver and heart fractions from up to eight mammalian species differing by 13-fold in longevity. Our results show, for the first time, a positive correlation between (mitochondrial) BER and mammalian longevity. This suggests that the low steady-state oxidative damage in mitochondrial DNA of long-lived species would be due to both their lower mitochondrial ROS generation and their higher mitochondrial BER. Long-lived mammals do not need to continuously maintain high nuclear BER levels because they release less mitROS to the cytosol. This can be the reason why they tend to show lower nuclear BER values. The higher mitochondrial BER of long-lived mammals contributes to their superior longevity, agrees with the updated version of the mitochondrial free radical theory of aging, and indicates the special relevance of mitochondria and mitROS for aging.

DNA repair in plant mitochondria - a complete base excision repair pathway in potato tuber mitochondria.

Mitochondria are one of the major sites of reactive oxygen species (ROS) production in the plant cell. ROS can damage DNA, and this damage is in many organisms mainly repaired by the base excision repair (BER) pathway. We know very little about DNA repair in plants especially in the mitochondria. Combining proteomics, bioinformatics, western blot and enzyme assays, we here demonstrate that the complete BER pathway is found in mitochondria isolated from potato (Solanum tuberosum) tubers. The enzyme activities of three DNA glycosylases and an apurinic/apyrimidinic (AP) endonuclease (APE) were characterized with respect to Mg2+ dependence and, in the case of the APE, temperature sensitivity. Evidence for the presence of the DNA polymerase and the DNA ligase, which complete the repair pathway by replacing the excised base and closing the gap, was also obtained. We tested the effect of oxidative stress on the mitochondrial BER pathway by incubating potato tubers under hypoxia. Protein carbonylation increased significantly in hypoxic tuber mitochondria indicative of increased oxidative stress. The activity of two BER enzymes increased significantly in response to this oxidative stress consistent with the role of the BER pathway in the repair of oxidative damage to mitochondrial DNA.

Estrés oxidativo mitocondrial y envejecimiento cardíaco / Mitochondrial oxidative stress and cardiac ageing

De acuerdo con diferentes organizaciones como la Asociación Americana del Corazón o la Organización Mundial de la Salud, las enfermedades cardiovasculares se han convertido en la primera causa de muerte en países occidentales. Aunque la exposición a diferentes factores de riesgo, en particular los relacionados con el estilo de vida, contribuyen de manera significativa a la etiopatogénesis de enfermedades cardíacas, el incremento en la esperanza de vida y el envejecimiento de la población asociado a él se consideran los determinantes principales del inicio y desarrollo de las mismas. Las mitocondrias y el estrés oxidativo se han señalado como factores relevantes tanto en el envejecimiento del corazón como en el desarrollo de enfermedades cardíacas como la insuficiencia cardíaca, la hipertrofia cardíaca y la miocardiopatía diabética. Durante el envejecimiento, diferentes procesos celulares relacionados con la función mitocondrial, como la bioenergética, procesos de apoptosis o de inflamación, se ven alterados, lo que conlleva una reducción en la supervivencia celular, y como consecuencia, disfunción cardíaca. Aumentar nuestro conocimiento sobre los mecanismos mitocondriales relacionados con el proceso de envejecimiento proporcionará nuevas estrategias para mejorar de forma más eficiente este proceso y las diferentes enfermedades relacionadas con él, en particular las cardiovasculares (AU)

According with different international organizations, cardiovascular diseases are becoming the first cause of death in western countries. Although exposure to different risk factors, particularly those related to lifestyle, contribute to the etiopathogenesis of cardiac disorders, the increase in average lifespan and aging are considered major determinants of cardiac diseases events. Mitochondria and oxidative stress have been pointed out as relevant factors both in heart aging and in the development of cardiac diseases such as heart failure, cardiac hypertrophy and diabetic cardiomyopathy. During aging, cellular processes related with mitochondrial function, such as bioenergetics, apoptosis and inflammation are altered leading to cardiac dysfunction. Increasing our knowledge about the mitochondrial mechanisms related with the aging process, will provide new strategies in order to improve this process, particularly the cardiovascular ones (AU)

Mitochondrial oxidative stress and cardiac ageing. / Estrés oxidativo mitocondrial y envejecimiento cardíaco.

According with different international organizations, cardiovascular diseases are becoming the first cause of death in western countries. Although exposure to different risk factors, particularly those related to lifestyle, contribute to the etiopathogenesis of cardiac disorders, the increase in average lifespan and aging are considered major determinants of cardiac diseases events. Mitochondria and oxidative stress have been pointed out as relevant factors both in heart aging and in the development of cardiac diseases such as heart failure, cardiac hypertrophy and diabetic cardiomyopathy. During aging, cellular processes related with mitochondrial function, such as bioenergetics, apoptosis and inflammation are altered leading to cardiac dysfunction. Increasing our knowledge about the mitochondrial mechanisms related with the aging process, will provide new strategies in order to improve this process, particularly the cardiovascular ones.

Role of Estrogen and Other Sex Hormones in Brain Aging. Neuroprotection and DNA Repair.

Aging is an inevitable biological process characterized by a progressive decline in physiological function and increased susceptibility to disease. The detrimental effects of aging are observed in all tissues, the brain being the most important one due to its main role in the homeostasis of the organism. As our knowledge about the underlying mechanisms of brain aging increases, potential approaches to preserve brain function rise significantly. Accumulating evidence suggests that loss of genomic maintenance may contribute to aging, especially in the central nervous system (CNS) owing to its low DNA repair capacity. Sex hormones, particularly estrogens, possess potent antioxidant properties and play important roles in maintaining normal reproductive and non-reproductive functions. They exert neuroprotective actions and their loss during aging and natural or surgical menopause is associated with mitochondrial dysfunction, neuroinflammation, synaptic decline, cognitive impairment and increased risk of age-related disorders. Moreover, loss of sex hormones has been suggested to promote an accelerated aging phenotype eventually leading to the development of brain hypometabolism, a feature often observed in menopausal women and prodromal Alzheimer's disease (AD). Although data on the relation between sex hormones and DNA repair mechanisms in the brain is still limited, various investigations have linked sex hormone levels with different DNA repair enzymes. Here, we review estrogen anti-aging and neuroprotective mechanisms, which are currently an area of intense study, together with the effect they may have on the DNA repair capacity in the brain.

Mitochondria and oxidative stress in heart aging.

As average lifespan of humans increases in western countries, cardiac diseases become the first cause of death. Aging is among the most important risk factors that increase susceptibility for developing cardiovascular diseases. The heart has very aerobic metabolism, and is highly dependent on mitochondrial function, since mitochondria generate more than 90 % of the intracellular ATP consumed by cardiomyocytes. In the last few decades, several investigations have supported the relevance of mitochondria and oxidative stress both in heart aging and in the development of cardiac diseases such as heart failure, cardiac hypertrophy, and diabetic cardiomyopathy. In the current review, we compile different studies corroborating this role. Increased mitochondria DNA instability, impaired bioenergetic efficiency, enhanced apoptosis, and inflammation processes are some of the events related to mitochondria that occur in aging heart, leading to reduced cellular survival and cardiac dysfunction. Knowing the mitochondrial mechanisms involved in the aging process will provide a better understanding of them and allow finding approaches to more efficiently improve this process.

Nuclear and mitochondrial DNA repair in selected eukaryotic aging model systems.

Knowledge about the different mechanisms underlying the aging process has increased exponentially in the last decades. The fact that the basic mechanisms involved in the aging process are believed to be universal allows the use of different model systems, from the simplest eukaryotic cells such as fungi to the most complex organisms such as mice or human. As our knowledge on the aging mechanisms in those model systems increases, our understanding of human aging and the potential interventions that we could approach rise significantly. Among the different mechanisms that have been implicated in the aging process, DNA repair is one of the processes which have been suggested to play an important role. Here, we review the latest investigations supporting the role of these mechanisms in the aging process, stressing how beneficial the use of different model systems is. We discuss how human genetic studies as well as several investigations on mammalian models and simpler eukaryotic organisms have contributed to a better understanding of the involvement of DNA repair mechanisms in aging.

Mitochondrial base excision repair assays.

Mitochondrial DNA (mtDNA) is constantly exposed to oxidative injury. Due to its location close to the main site of reactive oxygen species, the inner mitochondrial membrane, mtDNA is more susceptible than nuclear DNA to oxidative damage. The accumulation of DNA damage is thought to be particularly deleterious in post-mitotic cells, including neurons, and to play a critical role in the aging process and in a variety of diseases. Thus, efficient mtDNA repair is important for the maintenance of genomic integrity and a healthy life. The base excision repair (BER) mechanism was the first to be described in mitochondria, and consequently it is the best known. This chapter outlines protocols for isolating mitochondria from mammalian cells in culture and from rodent tissues including liver and brain. It also covers the isolation of synaptic mitochondria. BER takes place in four distinct steps, and protocols describing in vitro assays for measuring these enzymatic steps in lysates of isolated mitochondria are included.

Mitochondrial base excision repair in mouse synaptosomes during normal aging and in a model of Alzheimer's disease.

Brain aging is associated with synaptic decline and synaptic function is highly dependent on mitochondria. Increased levels of oxidative DNA base damage and accumulation of mitochondrial DNA (mtDNA) mutations or deletions lead to mitochondrial dysfunction, playing an important role in the aging process and the pathogenesis of several neurodegenerative diseases. Here we have investigated the repair of oxidative base damage, in synaptosomes of mouse brain during normal aging and in an AD model. During normal aging, a reduction in the base excision repair (BER) capacity was observed in the synaptosomal fraction, which was associated with a decrease in the level of BER proteins. However, we did not observe changes between the synaptosomal BER activities of presymptomatic and symptomatic AD mice harboring mutated amyolid precursor protein (APP), Tau, and presinilin-1 (PS1) (3xTgAD). Our findings suggest that the age-related reduction in BER capacity in the synaptosomal fraction might contribute to mitochondrial and synaptic dysfunction during aging. The development of AD-like pathology in the 3xTgAD mouse model was, however, not associated with deficiencies of the BER mechanisms in the synaptosomal fraction when the whole brain was analyzed.

Mitochondrial DNA repair and association with aging--an update.

Mitochondrial DNA is constantly exposed to oxidative injury. Due to its location close to the main site of reactive oxygen species, the inner mitochondrial membrane, mtDNA is more susceptible than nuclear DNA to oxidative damage. The accumulation of DNA damage is thought to play a critical role in the aging process and to be particularly deleterious in post-mitotic cells. Thus, DNA repair is an important mechanism for maintenance of genomic integrity. Despite the importance of mitochondria in the aging process, it was thought for many years that mitochondria lacked an enzymatic DNA repair system comparable to that in the nuclear compartment. However, it is now well established that DNA repair actively takes place in mitochondria. Oxidative DNA damage processing, base excision repair mechanisms were the first to be described in these organelles, and consequently the best understood. However, new proteins and novel DNA repair pathways, thought to be exclusively present in the nucleus, have recently been described also to be present in mitochondria. Here we review the main mitochondrial DNA repair pathways and their association with the aging process.

DNA damage and base excision repair in mitochondria and their role in aging.

During the last decades, our knowledge about the processes involved in the aging process has exponentially increased. However, further investigation will be still required to globally understand the complexity of aging. Aging is a multifactorial phenomenon characterized by increased susceptibility to cellular loss and functional decline, where mitochondrial DNA mutations and mitochondrial DNA damage response are thought to play important roles. Due to the proximity of mitochondrial DNA to the main sites of mitochondrial-free radical generation, oxidative stress is a major source of mitochondrial DNA mutations. Mitochondrial DNA repair mechanisms, in particular the base excision repair pathway, constitute an important mechanism for maintenance of mitochondrial DNA integrity. The results reviewed here support that mitochondrial DNA damage plays an important role in aging.

Differential age-related changes in mitochondrial DNA repair activities in mouse brain regions.

Aging in the brain is characterized by increased susceptibility to neuronal loss and functional decline, and mitochondrial DNA (mtDNA) mutations are thought to play an important role in these processes. Due to the proximity of mtDNA to the main sites of mitochondrial free radical generation, oxidative stress is a major source of DNA mutations in mitochondria. The base excision repair (BER) pathway removes oxidative lesions from mtDNA, thereby constituting an important mechanism to avoid accumulation of mtDNA mutations. The complexity of the brain implies that exposure and defence against oxidative stress varies among brain regions and hence some regions may be particularly prone to accumulation of mtDNA damages. In the current study we investigated the efficiency of the BER pathway throughout the murine lifespan in mitochondria from cortex and hippocampus, regions that are central in mammalian cognition, and which are severely affected during aging and in neurodegenerative diseases. A regional specific regulation of mitochondrial DNA repair activities was observed with aging. In cortical mitochondria, DNA glycosylase activities peaked at middle-age followed by a significant drop at old age. However, only minor changes were observed in hippocampal mitochondria during the whole lifespan of the animals. Furthermore, DNA glycosylase activities were lower in hippocampal than in cortical mitochondria. Mitochondrial AP endonuclease activity increased in old animals in both brain regions. Our data suggest an important regional specific regulation of mitochondrial BER during aging.

A potential impact of DNA repair on ageing and lifespan in the ageing model organism Podospora anserina: decrease in mitochondrial DNA repair activity during ageing.

The free radical theory of ageing states that ROS play a key role in age-related decrease in mitochondrial function via the damage of mitochondrial DNA (mtDNA), proteins and lipids. In the sexually reproducing ascomycete Podospora anserina ageing is, as in other eukaryotes, associated with mtDNA instability and mitochondrial dysfunction. Part of the mtDNA instabilities may arise due to accumulation of ROS induced mtDNA lesions, which, as previously suggested for mammals, may be caused by an age-related decrease in base excision repair (BER). Alignments of known BER protein sequences with the P. anserina genome revealed high homology. We report for the first time the presence of BER activities in P. anserina mitochondrial extracts. DNA glycosylase activities decrease with age, suggesting that the increased mtDNA instability with age may be caused by decreased ability to repair mtDNA damage and hence contribute to ageing and lifespan control in this ageing model. Additionally, we find low DNA glycosylase activities in the long-lived mutants grisea and DeltaPaCox17::ble, which are characterized by low mitochondrial ROS generation. Overall, our data identify a potential role of mtDNA repair in controlling ageing and life span in P. anserina, a mechanism possibly regulated in response to ROS levels.

Age-related cellular copper dynamics in the fungal ageing model Podospora anserina and in ageing human fibroblasts.

In previous investigations an impact of cellular copper homeostasis on ageing of the ascomycete Podospora anserina has been demonstrated. Here we provide new data indicating that mitochondria play a major role in this process. Determination of copper in the cytosolic fraction using total reflection X-ray fluorescence spectroscopy analysis and eGfp reporter gene studies indicate an age-related increase of cytosolic copper levels. We show that components of the mitochondrial matrix (i.e. eGFP targeted to mitochondria) become released from the organelle during ageing. Decreasing the accessibility of mitochondrial copper in P. anserina via targeting a copper metallothionein to the mitochondrial matrix was found to result in a switch from a copper-dependent cytochrome-c oxidase to a copper-independent alternative oxidase type of respiration and results in lifespan extension. In addition, we demonstrate that increased copper concentrations in the culture medium lead to the appearance of senescence biomarkers in human diploid fibroblasts (HDFs). Significantly, expression of copper-regulated genes is induced during in vitro ageing in medium devoid of excess copper suggesting that cytosolic copper levels also increase during senescence of HDFs. These data suggest that the identified molecular pathway of age-dependent copper dynamics may not be restricted to P. anserina but may be conserved from lower eukaryotes to humans.

Mitochondrial free radical generation and lifespan control in the fungal aging model Podospora anserina.

In the filamentous fungus Podospora anserina a central role of mitochondria in the control of aging has been repeatedly demonstrated. Interestingly, impairments in cytochrome c oxidase (COX) activity induce an enhancement in the expression of the quinol-oxygen alternative oxidoreductase (AOX) correlating with an extension of lifespan. This effect is thought to be determined by a reduction of the free radical generation in mitochondria. In the current investigation we have analyzed the electron transport chain composition of P. anserina and the superoxide generation rate in wild type s and in mutant grisea, a long-lived mutant with complex IV deficiency. Here we report that, similarly to other fungi, mitochondrial respiration in P. anserina is a combination of standard and alternative routes. A switch in the COX/AOX respiration balance affects the mitochondrial free radical generation. Lower mitochondrial rates of superoxide generation were found in the long-lived mutant, supporting the central role of mitochondrial free radical generation in the lifespan control of P. anserina. The question of how the activity of the alternative respiratory pathway influences the rate of free radical generation in P. anserina mitochondria is discussed.

Minireview: the role of oxidative stress in relation to caloric restriction and longevity.

Reduction of caloric intake without malnutrition is one of the most consistent experimental interventions that increases mean and maximum life spans in different species. For over 70 yr, caloric restriction has been studied, and during the last years the number of investigations on such nutritional intervention and aging has dramatically increased. Because caloric restriction decreases the aging rate, it constitutes an excellent approach to better understand the mechanisms underlying the aging process. Various investigations have reported reductions in steady-state oxidative damage to proteins, lipids, and DNA in animals subjected to restricted caloric intake. Most interestingly, several investigations have reported that these decreases in oxidative damage are related to a lowering of mitochondrial free radical generation rate in various tissues of the restricted animals. Thus, similar to what has been described for long-lived animals in comparative studies, a decrease in mitochondrial free radical generation has been suggested to be one of the main determinants of the extended life span observed in restricted animals. In this study we review recent reports of caloric restriction and longevity, focusing on mitochondrial oxidative stress and the proposed mechanisms leading to an extended longevity in calorie-restricted animals.

Dietary restriction at old age lowers mitochondrial oxygen radical production and leak at complex I and oxidative DNA damage in rat brain.

Previous studies in mammalian models indicate that the rate of mitochondrial reactive oxygen species ROS production and the ensuing modification of mitochondrial DNA (mtDNA) link oxidative stress to aging rate. However, there is scarce information concerning this in relation to caloric restriction (CR) in the brain, an organ of maximum relevance for ageing. Furthermore, it has never been studied if CR started late in life can improve those oxidative stress-related parameters. In this investigation, rats were subjected during 1 year to 40% CR starting at 24 months of age. This protocol of CR significantly decreased the rate of mitochondrial H(2)O(2) production (by 24%) and oxidative damage to mtDNA (by 23%) in the brain below the level of both old and young ad libitum-fed animals. In agreement with the progressive character of aging, the rate of H(2)O(2) production of brain mitochondria stayed constant with age. Oxidative damage to nuclear DNA increased with age and this increase was fully reversed by CR to the level of the young controls. The decrease in ROS production induced by CR was localized at Complex I and occurred without changes in oxygen consumption. Instead, the efficiency of brain mitochondria to avoid electron leak to oxygen at Complex I was increased by CR. The mechanism involved in that increase in efficiency was related to the degree of electronic reduction of the Complex I generator. The results agree with the idea that CR decreases aging rate in part by lowering the rate of free radical generation of mitochondria in the brain.

Short-term caloric restriction and sites of oxygen radical generation in kidney and skeletal muscle mitochondria.

Mitochondrial free radical generation is believed to be one of the principal factors determining aging rate, and complexes I and III have been described as the main sources of reactive oxygen species (ROS) within mitochondria in heart, brain, and liver. Moreover, complex I ROS generation of heart and liver mitochondria seems especially linked to aging rate both in comparative studies between animals with different longevities and in caloric restriction models. Caloric restriction (CR) is a well-documented manipulation that extends mean and maximum longevity. One of the factors that appears to be involved in such life span extension is the reduction in mitochondrial free radical generation at complex I. We have performed two parallel investigations, one studying the effect of short-term CR on oxygen radical generation in kidney and skeletal muscle (gastrocnemius) mitochondria and a second one regarding location of mitochondrial ROS-generating sites in these same tissues. In the former study, no effect of short-term caloric restriction was observed in mitochondrial free radical generation in either kidney or skeletal muscle. The latter study ruled out complex II as a principal source of free radicals in kidney and in skeletal muscle mitochondria, and, similar to previous investigations in heart and liver organelles, the main free radical generators were located at complexes I and III within the electron transport system.